In the early times of confocal raster light microscopy, which has the advantage that light emitted by parts of an object which are located outside the focal plane of the microscope objective is not blurring the image of the object of interest in the focal plane, so-called object scanner have been used, which move the respective object with regard to a static light beam. The construction of such an object scanner is described in U.S. Pat. No. 3,013,467. The use of an object scanner allows for a simple, static optic. Due to mechanical inertia, an object scanner, however, only allows for a comparatively low scanning velocity and correspondingly small frame rates. In biology and medicine, the scanning velocities which may be reached with an object scanner are only suitable for fixed dead samples.
Therefore, for imaging living cell structures in biological specimens, beam scanning methods are applied in confocal raster light microscopy, in which the light beam, generally a laser beam, is shifted with regard to an optic focussing the laser beam. Particularly, the light beam is shifted in such a way that it is pivoted about a fixed pivot point in the plane of the pupil of the focussing optic, which is here shortly referred to as the pupil of the focussing optic, to laterally shift the focus point of the light beam in the focal plane of the microscope objective. In microscope objectives of higher magnification, however, the pupil is located within the objective itself, and it is thus not mechanically accessible. Thus, the raster mechanism used has to be placed into an optical image of the pupil of the objective. If the beam pivot point is not exactly in the pupil of the focussing optic, the image brightness strongly drops towards the boundary of the scanning range, which is not acceptable.
Only one beam deflector, like for example a mirror, may be arranged in the pupil of the focussing optic or in each image of the pupil of the optic. For the purpose of scanning the scanning range in two orthogonal directions with a single mirror, it is known from DE 84 28 200 U1 to gimbal-mount the mirror in such a way that it is rotatable about the point of incident of the incident light beam in two orthogonal directions. The mechanical embodiment of this known raster scanner, however, does not allow for high scanning velocities, and the scanning precision is also limited.
Generally, it is possible to provide for two images of the pupil of the focussing optic and to arrange a beam deflector, which deflects the light beam in one of two orthogonal directions with regard to the focussing optic, in each of the two images of the pupil. In this way, however, the optical setup becomes complex and has negative effects on the optical transmission.
If one of two separate rotating mirrors, which are rotatable about orthogonal pivot axes, or if even both of them are not arranged in but only close to a single image of the pupil of the focussing optic, this results in a geometric distortion and aberrations besides a varying image brightness over the scanning range.
An overview over known laser scanners is provided by James B. Pawley: “Handbook of Biological Confocal Microscopy”, 3rd edition, Springer Verlag, ISBN 10: 0-387-25921-X, ISBN 13: 987-0387-25921-5.
From DE 40 26 130 A1 an apparatus for and a method of dynamically shifting a light beam with regard to an optic focussing the light beam are known, in which a laser beam is deflected in one direction by two rotating mirrors whose rotating movements about parallel pivot axes are coupled according to a fixed mathematical relation in such a way that the laser beam is pivoted about a fixed point of incident on a further rotating mirror. By means of rotating this further rotating mirror, the laser beam is deflected in the second direction. If the point of incidenceis in the entrance pupil of the focussing optic, the common pivot point of the laser beam in both directions falls in the pupil of the focussing optic.
A further apparatus for and a further method of dynamically shifting a light beam with regard to an optic focussing the light beam are known from DE 196 54 210 A1. Here, two mirrors which are fixed with regard to each other in a predetermined relative angle position are provided for deflecting the light beam in a first direction. Thus, these two mirrors are simultaneously rotatable about the optical axis of the incident light beam in such a way that the light beam falls in a fixed point onto a further rotating mirror which is arranged on the extended optical axis of the incident light beam, and which is rotatable about a pivot axis running orthogonally both to the incident light beam and to the deflected light beam. Due to the arrangement of the further rotating mirror in an image of the pupil of the focussing optic, the light beam is pivoted about a single point in the pupil image in both directions. In the known apparatus, further mirrors may be provided to pivot the focussed light beam about the optical axis of the incident light beam.
In recent time, methods of high resolution raster light microscopy have been developed, which achieve spatial resolutions beyond the diffraction barrier. One example for these high resolution methods is STED (Stimulated Emission Depletion) microscopy as described in WO 95/21393 A. The new high resolution methods place much higher demands on imaging the light beam, by which the scanning range is scanned, into the pupil of a focussing optic than common raster light microscopy. For example, in STED microscopy, a first fluorescence excitation light beam is used for fluorescence excitation like in confocal raster light microscopy. Additionally, however, a second stimulation light beam is used, which avoids the occurrence of fluorescence in the boundary of the diffraction-delimited spot of the focused excitation light beam. For this purpose, the intensity distribution of the stimulation light in the focal plane of the focussing optic is made ring-shaped, for example. To provide an intensity zero point in the center of the intensity distribution of the stimulation light, i. e. at the focus point of the excitation light, the electrical field of the stimulation light beam is spatially modulated in an image of the pupil of the objective by means of so-called phase plates or phase modulators for example in such a way that the integral of the electric field over the pupil becomes zero. In practical applications of STED microscopy, the remaining intensity of the stimulation light in the zero point of the intensity distribution has to be smaller than one percent of the maximum ambient light intensity to not also eliminate the measurement signal of interest from the focus point of the excitation light. As a result, in scanning the scanning range by deflecting the excitation and the stimulation light beam, the spatial phase structure of the stimulation light beam in the pupil of the focussing optic may only be moved by a very small fraction of the pupil diameter, for certain phase plates by only about one thousandth of the pupil diameter at maximum. With known light scanner types, this criterion, if met at all, is only met with an extreme effort, i.e. with highest-value optical components for the rotating mirrors and for the focussing optic.
From US 2006/0151449 A1 a system and a method for scanning a surface with a collimated beam are known. The collimated light beam, for example a laser beam, is laterally shifted in parallel to its optical axis by means of two pairs of beam deflectors, such as mirrors. The beam deflectors are arranged in series along the beam path, each of them being rotatable about one of the following axes: two pivot axes running in parallel to each other and perpendicular to the light beam and two further pivot axes running in parallel to each other and perpendicular to the light beam but also perpendicular to the first two axes. The two beam deflectors of each pair of beam deflectors which are rotatable about parallel pivot axes are both pivoted by same angles in the same direction to provide for a parallel shift of the laser beam. Thus, for example, rotating mirrors as beam deflectors are arranged in pairs whose surface normals are anti-parallel in each operation position of the known system. The laser beam is not focused in this known system and method.
There still is a need for an apparatus for and a method of dynamically shifting a light beam with regard to an optic focussing the light beam, by which, without high practical or constructional effort, it is possible to pivot the light beam about a fixed pivot point in the pupil of the focussing optic in two directions to scan a two-dimensional scanning range without variations of the optical conditions over the scanning range.